Project Summary/Abstract The mitochondrial pathway of apoptosis is the major mechanism whereby cells in vertebrate organisms undergo programmed cell death. It is vital for homeostasis of the immune system, tumor suppression and the regulation of cell number in development. Mitochondrial outer membrane permeabilization (MOMP) and release of intermembrane space proteins such as cytochrome C are central to this process. MOMP is controlled by the Bcl-2 family of proteins. Pro-apoptotic members, Bax and Bak, are activated by another subgroup of the Bcl-2 family, BH3-only proteins, and induce the release of intermembrane space proteins. Anti-apoptotic members of the Bcl-2 family inhibit this process. Based on the finding that BH3-only proteins bind to anti-apoptotic Bcl-2 family members to induce apoptosis, drugs that mimic this effect were developed and are being used in cancer therapy. Better understanding of how Bcl-2 family proteins interact with the MOM and induce permeabilization will allow generation of new or even better drugs. The central hypothesis of this proposal is that there are MOM proteins that co- operate with pro-apoptotic Bcl-2 family proteins to generate lipidic pores in the MOM. The object of this application is to identify these molecules and to determine how they contribute to Bax and Bak activation followed by pore formation. Our Specific Aims are: Aim 1. Identify MOM proteins that co-operate with Bid for induction of MOMP. Unknown MOM proteins assist Bid in Bax mediated-permeabilization of membranes. MOM proteins will be fractionated and reconstituted in proteo-liposomes to identify the active fraction by the dextran release assay. We will investigate whether the identified MOM protein knockdown has the same inhibitory effect as Bid deficiency in mouse embryonic fibroblast (MEF) lines. Aim 2. Determine how Bax activation is regulated prior to MOMP. Using a dextran release kinetics assay, we will determine the time course of Bax conformational change and oligomerization in relation to membrane permeabilization. We will also determine the mechanism of Bcl-xL inhibition by analysis of its effect on release kinetics. We will follow up these experiments by examining cytochrome C release kinetics using mitochondria isolated from cytochrome C-GFP expressing cells in time lapse fluorescent microscopy. Aim 3. Demonstrate lipidic pores induced by Bax and Bak and determine whether Bax localizes to these pores. Our cryo-EM data suggest that Bax generates lipidic pores in the membrane. We will use high-pressure freezing/freeze substitution-EM to demonstrate the existence of these pores in OMVs and mitochondria both in isolation and in cells. This technique will allow us to detect pores with immunogold or nanogold labeled Bax. Mapping the distribution of Bax along the edge of the pores will enable us to determine whether Bax pores are mainly lipidic or proteinaceous.